Hedgehog signaling has been implicated in many cancers and has been shown to be a useful target for anti-cancer therapy. Previous work, including key contributions from my lab, have established that the Hedgehog protein is one of the major regulators of animal development, playing key roles in most tissues and organs in both invertebrates and vertebrates. Using newly developed reagents and methods that open new ways to study Hh signaling, the work proposed in this application will identify and characterize the processes that Hh-producing cells use to prepare and deliver Hh protein, the processes that Hh-receiving cells use to prepare for and handle its arrival, and the processes that regulate the form and activity of Ci, the transcription factor that is responsible for Hh pathway output. These issues are critical to understanding how new and better therapeutic regimens controlling Hh activity can be developed.